The present invention relates to cancer control. More specifically, it relates to a method for controlling cancer cell invasion which utilizes control of the matrix metalloproteinase transcription-activating function of E1AF gene, and use thereof in a method for detecting cancer cells, a method for diagnosing cancer invasion and a kit for carrying out these methods.
The present inventors succeeded in isolation of E1AF gene encoding E1AF protein, which binds to the enhancer region of E1A gene of adenovirus type 5, from human cell strain HeLa [JP 5-328975 A: Nucleic Acids Research, 21, 547-553 (1993)]. The nucleotide sequence of E1AF gene and the amino acid sequence of E1AF protein expressed by the gene are shown in the Sequence Listing, SEQ ID NO 1, hereinafter.
E1AF gene is a new oncogene belonging to ets group oncogenes (ets family oncogenes) and it has been shown to be human homolog of PEA 3 isolated from mouse [Xin et al., Genes Dev., 6, 481-496 (1992)].
About 30 proteins expressed by ets family oncogenes have been found. They have a common structure called as ETS domain and this structure has DNA-binding activity. It has been considered that proteins expressed by ets family oncogenes function as transcription factors and play important roles in control of gene expression upon cell growth, transformation and the like [Waslylyk et al., Eur. J. Biochem., 211, 7-18 (1993)].
However, the functions of E1AF gene are unclear.
Objects of the present invention are to clarify the functions of E1AF gene, in particular, the relationship between activation of matrix metalloproteinases and the invasion and metastasis of cancers, and to provide a method for controlling E1AF gene, thereby controlling the invasion and metastasis of cancers, a method for detecting expression of E1AF gene, thereby evaluating malignancy of cancers and a means for carrying out the methods.
The present inventors have studied the relationship between E1AF gene and matrix metalloproteinases or E1AF gene and the invasion and metastasis of cancers, intensively. As a result, it has been found that E1AF protein expressed by E1AF gene acts on promoters of various matrix metalloproteinase genes to increase their promoter activities, remarkably, thereby enhancing cancer cell invasion. In addition, the present inventors have found that cancer cell invasion can be controlled, if expression of E1AF gene is controlled by using genetic engineering techniques. Furthermore, the present inventors have found that cancer cell invasion can be controlled, if a DNA-binding domain (ETS domain) of E1AF protein is transferred to cancer cells by genetic engineering techniques. Moreover, the present inventors have found methods for detecting cancers and diagnosing cancer invasion by utilizing the expression of E1AF gene as an index. Thus, the present invention has been completed.
That is, briefly, the first aspect of the present invention relates to a method for controlling cancer cell invasion and is characterized by controlling the matrix metalloproteinase-transcription-activating function of E1AF gene, in particular, controlling E1AF or its expression product, or its functional equivalent. The second aspect of the present invention relates to a method for detecting cancers and is characterized by detecting a product expressed by E1AF gene. The third aspect of the present invention relates to a method for diagnosing cancer tissue invasion and is characterized by detecting a product expressed by E1AF gene in cancer tissue isolated from human being. The fourth aspect of the present invention relates to a kit for controlling cancer cell invasion and is characterized by comprising as a constituent component a material for controlling the matrix metalloproteinase transcription-activating function of E1AF gene. The fifth aspect of the present invention relates to a kit for detecting cancers and is characterized by comprising, as a constituent component, a probe which is hybridizable to mRNA of E1AF gene.
The wording xe2x80x9cfunctional equivalentxe2x80x9d used herein means as follows:
In naturally occurring proteins, in addition to polymorphism or mutation of genes encoding the proteins, mutations such as deletion, substitution, insertion and/or addition of amino acid(s) in their amino acid sequences may occur owing to, for example, modification reactions in a living body and during purification after formation of the proteins. It has been known that there are some proteins which maintain substantially the same physiological and biological activities as those of the original proteins, even after undergoing such mutation. Thus, in case that any significant difference is not found, even when there is a structural difference, it is called a xe2x80x9cfunctional equivalentxe2x80x9d herein.
Even when the above mutation is introduced into the amino acid sequence of protein artificially, the situation is unchanged. In this case, many more variants can be produced and, in so far as substantially the same physiological activities are maintained, the variants are recognized to be included in functional equivalents.
For example, in many cases, it is said that methionine residue present in the N-terminal of protein expressed in E. coli is removed by the action of methionine aminopeptidase and both products with and without methionine residue are formed according to a particular kind of proteins. However, in many cases, this presence of methionine residue does not influence the activities of proteins. In addition, it has been known that in a polypeptide whose amino acid sequence is similar to human interleukin 2 (IL-2) but wherein a certain cysteine residue has been replaced with serine maintains IL-2 activity [Science, 224, 1431 (1984)].
Furthermore, when proteins are produced by gene engineering techniques, they are often expressed as fusion proteins. For example, an N-terminal peptide chain derived from different protein is added to the N-terminal of the desired protein to increase the amount of the expression of the desired protein; or the desired protein is expressed with addition of a suitable peptide chain to the N- or C-terminal thereof and a carrier having affinity to the peptide chain added thereto is used to facilitate purification of the desired protein.
In addition, it has been known that, for each amino acid, there are 1 to 6 codons which represent the same amino acid on a gene (combination of three nucleotides). Then, many genes encoding one particular amino acid sequence can exist, though it depends on the kind of the amino acid. Genes are by no means present stably in nature and mutation of their nucleotide sequences often occur. There are cases where mutation of a gene does not cause any change in the amino acid sequence encoded by the gene (sometimes called as silent mutation). In such cases, it is considered that different genes encoding the same amino acid sequence are formed. Then, even if a gene encoding a certain amino acid sequence has been isolated, there is a possibility that various kinds of genes encoding the same amino acid sequence are formed during passage of an organism containing the gene.
Moreover, various genes which encode the same amino acid sequence can be produced without any difficulty by utilizing various genetic engineering techniques.
For example, in the production of protein by a genetic engineering technique, when a codon used in an inherent gene encoding the desired protein has a low frequency in a host used, sometimes, only a small amount of the protein is expressed. In such a case, for increasing in the amount of the desired protein expressed, the codon is changed to another one which is frequently used in the host artificially without changing the amino acid sequence to be encoded. Needless to say, in this way, many genes which encode a specific amino acid sequence can be produced artificially. Then, even if nucleotide sequences of genes produced artificially have different nucleotide sequences, in so far as they encode the amino acid sequences as disclosed herein, they are included in the present invention.
Further, in many cases, polypeptides having deletion, substitution, insertion and/or addition of one or plural amino acids in the amino acid sequence of the desired protein have the same functional activity as that of the desired protein. Genes encoding such polypeptides are also included in the present invention regardless of their being naturally occurring genes or artificial genes.
In general, for the functional equivalent, there are many cases where genes encode materials having homology to each other. Therefore, genes which can hybridize to the genes of the present invention and function in the same way are also included in the present invention.
The wording xe2x80x9ctarget DNAxe2x80x9d used herein means DNA that binds to a DNA binding domain of E1AF protein to inhibit the matrix metalloproteinase transcription-activating function of E1AF gene. Preferably, it means the following DNA.
It has been known that DNA which binds to a DNA binding domain of E1AF protein is 5xe2x80x2-MGGAWGT-3xe2x80x2 (wherein M represents A or C and W represents A or T) [F. Higashino et al., Nucleic Acids Research, 21, 547-553 (1933)]. In addition, it has been revealed that a DNA conserved sequence to which proteins of ets family oncogenes including E1AF protein bind is 5xe2x80x2-MGGAW-3xe2x80x2 [B. Wasylyk et al., Eur. J. Biochem., 211, 7-18 (1993)]. Furthermore, the promoters or enhancer regions of matrix metalloproteinases whose transcription is activated by E1AF protein have DNA binding sequences to which the E1AF protein binds. That is, E1AF binding sequence is found in xe2x88x9289 region of Type I collagenase, xe2x88x92216 and xe2x88x92208 regions of stromelysin, xe2x88x92181 region of stromelysin 2 and xe2x88x92170 and xe2x88x92146 regions of matrilysin [M. Gaire et al., J. Biol. Chem., 269, 2032-2040 (1994)]. Moreover, E1AF binding sequence is present in xe2x88x92541 region (5xe2x80x2-AGGAAG3xe2x80x2) and xe2x88x92230 region [5xe2x80x2-AGGAAA-3xe2x80x2, this is oriented in the reverse of the enzyme gene (hereinafter referred to as minus strand)] of 92 kD Type IV collagenase [P. Huhtala et al., J. Biol., Chem., 266, 16845-16849 (1991)]. E1AF binding sequence is also present in xe2x88x92388 region (5xe2x80x2-AGGAAT-3xe2x80x2, minus strand), xe2x88x92378 region (5xe2x80x2-AGGAAC-3xe2x80x2, minus strand) and xe2x88x92144 region (5xe2x80x2AGGAAA-3xe2x80x2) of 72 kD Type IV collagenase [P. Huhtala et al., J. Biol. Chem., 265, 11077-11082 (1990)].
Thus, any DNA which has the 5xe2x80x2-MGGAW-3xe2x80x2 sequence and inhibits the matrix metalloproteinase transcription-activating function of E1AF gene is included in the target DNA used herein.
The term xe2x80x9cdecoyxe2x80x9d used herein means transcription factor-binding protein and DNA having a sequence of transcription factor-binding site or a similar sequence and is a material which inhibits the function of a transcription factor by introducing it into cells as a decoy. In particular, the decoy is a material which inhibits the matrix metalloproteinase transcription-activating function of E1AF gene. For example, a DNA binding domain of E1AF protein, a functional equivalent of a DNA binding domain or target DNA of a DNA binding domain of E1AF protein can be used as the decoy. In addition, a DNA binding domain of E1AF protein obtained by using a genetic engineering technique or an expression product of a gene encoding a functional equivalent of the DNA binding domain can be used as the decoy.
A material which can effectively inhibit the matrix metalloproteinase-transcription-activating function of E1AF gene is included in the decoy of the present invention.
Further, the term xe2x80x9cribozymexe2x80x9d used herein is an enzyme which degrades mRNA of E1AF protein or mRNA of matrix metalloproteinase protein, thereby inhibiting translation into the protein to inhibit cancer cell invasion. For example, hammer-head type ribozyme 5xe2x80x2-NNNNNNNCUGAUGAAGGGUGAUACCCUGAAAC (or G) Nxe2x80x2Nxe2x80x2Nxe2x80x2Nxe2x80x2Nxe2x80x2-3xe2x80x2 (SEQ ID NO 2, N and Nxe2x80x2 represent any nucleotide and the sequences form a hybrid with a target mRNA) can be used [M. Koizumi et al., FEBS Letter, 228, 228-230(1988)]. In this case, as the target mRNA, the sequence of 5xe2x80x2-Nxe2x80x2Nxe2x80x2Nxe2x80x2Nxe2x80x2Nxe2x80x2G (or, C, when the hammer-head type ribozyme is G ) UHNNNNNNN-3xe2x80x2 (SEQ ID NO 3, N and Nxe2x80x2 represent any nucleotide, H is A, C or U provided that N and Nxe2x80x2 are the sequences which form a hybrid with the above N and Nxe2x80x2 of the hammer-head type ribozyme) is required. That is, the presence of the sequence, 5xe2x80x2-G (or C) UH-3xe2x80x2, is required.
Ribozymes designed from these sequences are also included in the ribozyme used herein. In addition, the ribozyme used herein includes any ribozyme which can break mRNA of E1AF protein or mRNA of a matrix metalloproteinase and inhibit translation into the protein to control cancer cell invasion regardless of its kind such as another hammer-head type ribozyme, hairpin type ribozyme, delta type ribozyme or the like.
The invasion and metastasis of cancers can be controlled by enhancing expression of E1AF gene. CAT assay which is used for the analysis of promoters can be employed for investigating whether E1AF protein expressed by E1AF gene can activate promoters of various matrix metalloproteinases. That is, a plasmid is constructed by inserting the promoter region of a matrix metalloproteinase into the upstream from a chloramphenicol acetyltransferase (CAT) reporter gene. Then, cells are transfected with the plasmid together with an E1AF gene expression vector and CAT activity is measured to determine the activation of the promoter of the matrix metalloproteinase.
For investigating the relationship between E1AF gene and cancer cell invasion, a cancer cell line having a low invasion capability transfected with an E1AF gene expression vector is subjected to Matrigel assay method to evaluate the change of in vitro invasion. In addition, the invasion and metastasis can be determined by animal experiments.
The invasion and metastasis of cancers can be controlled by inhibiting the expression of E1AF gene. As a method for controlling the expression of E1AF gene, anti-sense RNA, anti-sense DNA and triple helix techniques and the like can be utilized. And, by using a decoy, the invasion and metastasis of cancers can be controlled. As the decoy, a DNA binding domain of E1AF protein, a functional equivalent of the DNA binding domain, an expression product by a gene encoding the DNA binding domain of E1AF protein, an expression product by a gene encoding a functional equivalent of the DNA binding domain and target DNA of the DNA binding domain of E1AF protein can be used.
The detection of cancers and the diagnosis of cancer invasion can be carried out by monitoring the expression of E1AF gene as an index. For investigating the expression of E1AF gene, RNA can be prepared from cancer tissue to determine the expression level of mRNA by northern hybridization or PCR. And, regarding the expression amount of E1AF protein, an antibody for cancer tissue against E1AF protein can be used.
Thus, in the method for controlling cancer cell invasion of the present invention, the matrix metalloproteinase transcription-activating function of E1AF gene is controlled.
When the matrix metalloproteinase-transcription-activating function is enhanced, cancer cell invasion is activated. On the other hand, when it is inhibited, cancer cell invasion is inhibited.
Cancer cell invasion can be activated by introducing a material for enhancing the matrix metalloproteinase-transcription-activating function of E1AF gene, for example, introducing E1AF gene into the cells so that the gene is expressed in the cells.
E1AF gene represented by SEQ ID NO 1 of the Sequence Listing or a part thereof can be introduced in the form of a vector so that the gene is maintained extrachromosomally. In such a state, the gene is expressed from an extrachromosomal position by a cell. When a part of E1AF gene is introduced into a cell and is expressed, the introduced part of the gene should encode a necessary part for activating cell invasion. A vector for maintaining a gene extrachromosomally has been known in the art and an appropriate vector is used. As a method for introducing a DNA into a cell, for example, electroporation, calcium phosphate co-precipitation, virus transduction and the like have been known and the choice of a particular method is within routine work for a skilled artisan.
Further, E1AF gene or a part thereof can be introduced in the form of a vector so that the gene is integrated intrachromosomally. In such a state, the gene is maintained intrachromosomally and expressed by a cell. A viral vector can be used as a vector for integrating the gene intrachromosomally to introduce the gene into cancer cells efficiently. As the vector, retrovirus, vaccinia virus, adeonvirus, a non-proliferative recombinant virus or the like can be used.
Cancer cells wherein E1AF gene has been expressed are useful as a model system for studying cell invasion or a model system for studying therapeutic drugs for inhibiting cancer cell invasion. In addition, they are also useful as a model system for studying cancer cell metastasis or a model system for studying medicament for inhibiting metastasis of cancers. Cells to be used as the model systems include, for example, human breast cancer cells, human osteosarcoma cells and the like. After the expression of E1AF gene in cancer cells, a test material is applied to the cancer cells and the metastasis inhibitory activity of a test material is evaluated based on invasiveness and motility of the cells, metastasis activity in a nude mouse and the like. If a test material inhibits metastasis of cancer, the material is a candidate for a drug for inhibiting metastasis of cancers such as breast cancer, osteosarcoma and the like.
Cancer cell invasion is activated by introducing E1AF protein expressed by E1AF gene or its functional equivalent into cancer cells. The sequence of the expressed E1AF protein is shown as SEQ ID NO:5 of the Sequence Listing.
E1AF protein or its functional equivalent can be produced by, for example, using a known vector to express cDNA in a microorganism. Alternatively, it can be extracted from cells producing E1AF protein or its functional equivalent. In addition, E1AF protein or its functional equivalent can be synthesized by synthetic chemical techniques. Any of these methods can provide a preparation containing E1AF protein or its functional equivalent of the present invention. This preparation is substantially free from other human proteins. This can be most readily accomplished by synthesizing in a microorganism or in vitro.
E1AF protein or its functional equivalent can be introduced into cells by using microinjection, liposomes or the like. Alternatively, the protein or its functional equivalent can be introduced into cells by a drug delivery system or diffusion. Activation of the invasion and metastasis of cancer cells can be evaluated by determining the invasiveness motility of cancer cells in which E1AF protein or its functional equivalent is introduced and their metastasis in an animal model. Cancer cell invasion is activated by introducing E1AF protein or its functional equivalent.
The activated cancer cells are useful as a model system for studying cancer invasion, a model system for studying cancer metastasis and development of cancer invasion inhibitory drugs and cancer metastasis inhibitory drugs.
On the other hand, cancer cell invasion is inhibited by introducing a material inhibiting the matrix metalloproteinase-transcription-activating function of E1AF gene, for example, anti-sense DNA or anti-sense RNA of E1AF gene.
As the anti-sense DNA to be used for the introduction of anti-sense DNA of E1AF gene or a part thereof can be used. As the anti-sense RNA to be used, anti-sense RNA or a part thereof can be used. Any anti-sense DNA or anti-sense RNA can be used in so far as it can be hybridized with mRNA of E1AF gene in a cell after introduction into the cell to inhibit the function of the gene. These anti-sense DNA and anti-sense RNA can be prepared by synthetic chemical methods or genetic engineering techniques and the like. The anti-sense DNA and anti-sense RNA can be introduced into cells by microinjection, polyethylene glycol method, particle method, electroporation, liposome method and the like.
The anti-sense RNA can be expressed in cancer cells by integrating E1AF gene or a part thereof into the above vector for maintaining a gene extrachromosomally or the above vector for integrating a gene intrachromosomally and introduced cancer cells to express the anti-sense RNA in the cells. An RNA expression vector of the anti-sense RNA can be introduced into cells by electroporation, calcium phosphate co-precipitation, viral transduction and the like.
The effect of the length of the anti-sense DNA or anti-sense RNA and its sequence on inhibition can be evaluated by determining cancer cell invasion after introduction of the anti-sense DNA or the anti-sense RNA. Cancer cells in which E1AF gene expression is inhibited by anti-sense DNA or anti-sense RNA are useful as a model system for studying invasiveness and motility and metastasis of cancer cells.
Cancer cell invasion can be inhibited by introducing a DNA binding domain of E1AF protein or its functional equivalent.
As the DNA binding domain of E1AF protein or its functional equivalent, any of such materials can be used in so far as it can bind to target DNA relating to cancer cell invasion to inhibit the matrix metalloproteinase transcription-activating function of intracellular E1AF protein. Preferably, the material has no transcription-activating function and no acidic amino acid region. The DNA binding domain of E1AF protein is represented by the amino acid sequence of from the 315th to 399th amino acids in SEQ ID No 5: of the Sequence Listing.
The DNA binding domain of E1AF protein or its functional equivalent can be produced by, for example, using a known expression vector to express cDNA in a microorganism. Alternatively, the DNA binding domain of E1AF protein or its functional equivalent can be synthesized by using synthetic chemical techniques. Any of these techniques can be used for providing a preparation containing the DNA binding domain of E1AF protein or its functional equivalent of the present invention. The preparation is substantially free from any other human protein. This can be most readily accomplished by synthesizing in a microorganism or in vitro.
The DNA binding domain of E1AF protein or its functional equivalent can be introduced into cells by utilizing microinjection, liposome and the like. Alternatively, the domain or its functional equivalent can be introduced into cells by a drug delivery system or diffusion.
The inhibition of cancer cell invasion and metastasis can be evaluated by determining invasiveness and mobility of cancer cells into which a DNA binding domain of E1AF protein or its functional equivalent is introduced and by determining metastasis in an animal model. The activation of cancer cell invasion by E1AF protein is inhibited by introduction of a DNA binding domain of E1AF protein or its functional equivalent into cells.
Cancer cell invasion can be inhibited by introducing a gene encoding a DNA binding domain of E1AF protein or a gene encoding a functional equivalent of a DNA binding domain of E1AF protein into cells so that the gene is expressed in the cells. Into cells are introduced a gene encoding the DNA binding domain of E1AF protein represented by the amino acid sequence of from the 315th to 399th amino acids of SEQ ID NO 5 of the Sequence Listing or a gene containing a part thereof, or a gene encoding a functional equivalent of the DNA binding domain of E1AF protein or a gene containing a part thereof in the form of a vector so that the gene stays extrachromosomally. In such a state, the gene is expressed from an extrachromosomal position by the cells. When a part of a gene encoding the DNA binding domain of E1AF protein or a part of a gene encoding a functional equivalent of the DNA binding domain of E1AF protein to express the gene are introduced into cells, the part of the gene to be introduced into the cells should contain a part encoding the DNA binding domain or the functional equivalent required for inhibiting cell invasion. A vector for maintaining a gene extrachromosomally has been known in the art and an appropriate vector is used. A method for introduction of DNA into cells has been known and, for example, there are electroporation, calcium phosphate co-precipitation, virus transduction and the like. The choice of a particular method is within routine work for a skilled artisan.
Further, a gene encoding the DNA binding domain of E1AF protein or a part thereof, or a gene encoding the functional equivalent of the DNA binding domain of E1AF protein or a part thereof in the form of a vector can be introduced into cells so that the gene is integrated intrachromosomally. In such a state, the gene is maintained intrachromosomally and expressed by the cells. A viral vector can be used as a vector for introduction of the gene intrachromosomally. As the vector, retrovirus, vaccinia virus, adeonvirus, a non-proliferative recombinant virus, etc. can be used.
Cancer cell invasion can also be inhibited by introduction of a target DNA of the DNA binding domain of E1AF protein into cells.
As the target DNA of the DNA binding domain of E1AF protein, any one containing DNA which can binds to the DNA binding domain of E1AF protein can be used. In particular, a DNA containing the sequence of 5xe2x80x2-MGGAW-3xe2x80x2 can-be used. As the DNA, a promoter or enhancer region containing a binding sequence of a matrix metalloproteinase gene which binds to the DNA binding domain of E1AF protein can be used. The DNA to be used may be that extracted and purified from a microorganism, after amplification of the DNA in the microorganism by using a known vector, or the DNA can be amplified in vitro by PCR or the like. In addition, synthetic DNA can be used. The target DNA obtained by these methods according to the above various methods or a vector so that the DNA stays extrachromosomally can be introduced into cancer cells. In such a state, the DNA is amplified from an extrachromosomal position by the cells. A vector for maintaining the DNA extrachromosomally has been known in the art and an appropriate vector is used. As a method for introducing DNA into cells, for example, electroporation, calcium phosphate coprecipitation, virus transduction and the like have been known and the choice of a particular method is within routine work for a skilled artisan.
Cancer cell invasion can be inhibited by introducing ribozyme for mRNA of E1AF gene into cancer cells.
The ribozyme should be designed so that it functions in cancer cells stably. When the ribozyme is a hammer-head type, 5xe2x80x2-GUC-3xe2x80x2 (SEQ ID NO 1, 107th-109th nucleotides) is present in E1AF gene. The ribozyme is designed and synthesized from this sequence. The synthesized ribozyme by the above various methods can be introduced into cancer cells or the ribozyme can be inserted into the above vector and introduced into cancer cells to express in the cells extrachromosomally or intrachromosomally.
Cancer cell invasion can be inhibited by introducing ribozyme for mRNA of a matrix metalloproteinase gene into cancer cells.
The ribozyme should be designed so that it functions in cancer cells stably. In case that the ribozyme is a hammer-head type, 5xe2x80x2-GUC-3xe2x80x2 (114th-116th nucleotides) is present in Type I collagenase [P. Angel et al., Mol. Cell. Biol., 7, 2256-2266(1987)], 5xe2x80x2-GUC-3xe2x80x2(51st-53rd nucleotides) is present in stromelysin tS.E. Whitham et al., Biochem. J., 240, 913-916 (1986)], 5xe2x80x2-GUU-3xe2x80x2 (49th-51st nucleotides) is present in stromelysin 2 [D. Muller et al., Biochem. J., 253, 187-192 (1988)], 5xe2x80x2-GUC-3xe2x80x2 (41st-43rd nucleotides) is present in 92 kD Type IV collagenase [P. Huhtala et al., J. Biol. Chem., 266, 16845-16849 (1991)], and 5xe2x80x2-GUC-3xe2x80x2 (342nd-344th nucleotides) is present in 72 kD Type IV collagenase [P. Huhtala et al., J. biol., Chem., 265, 11077-11082 (1990)]. The ribozyme is designed and synthesized based on this sequence. The synthesized ribozyme can be introduced into dancer cells by the above various method or inserted into the above vector and introduced into cancer cells to express in the cells extrachromosomally or intrachromosomally.
Cancer cell invasion can be inhibited by using anti-sense DNA or anti-sense RNA of E1AF gene, ribozyme for mRNA of E1AF gene and ribozyme for mRNA of a matrix metalloproteinase gene and these materials are useful as cancer-invasion-inhibiting drugs.
In addition, cancer cell invasion can be inhibited by using, as decoy type drugs, a DNA binding domain of E1AF protein, a functional equivalent of a DNA binding domain of E1AF protein, a product expressed by a gene encoding a DNA binding domain of E1AF protein or a functional equivalent of a DNA binding domain of E1AF protein and target DNA of a DNA binding domain of E1AF protein. These entities are also useful as cancer-invasion-inhibiting drugs.
For example, anti-sense DNA, anti-sense RNA, an expression vector of anti-sense RNA, a DNA binding domain of E1AF protein, a functional equivalent of a DNA binding domain of E1AF protein, an expression vector of a DNA binding domain of E1AF protein, an expression vector of a functional equivalent of a DNA binding domain of E1AF protein, target DNA of a DNA binding domain of E1AF protein, an expression vector of target DNA of a DNA binding domain of E1AF protein, ribozyme for mRNA of E1AF gene, an expression vector of ribozyme for mRNA of E1AF gene, ribozyme for mRNA of a matrix metalloproteinase gene or an expression vector of ribozyme for mRNA of a matrix metalloproteinase gene can be injected into the diseased site on the surface of tissue, directly, and can be injected into cancer cells and surrounding tissue to inhibit cancer metastasis efficiently. Further, they can be injected into the disease site at the inside of tissue and surrounding tissue, directly. Alternatively, a drug delivery system can be used. As the drug delivery system, systems specific to cancer cells are preferred and can be selected from general systems utilizing cancer cell receptors, cancer cell specific antibodies and the like.
Thus, anti-sense DNA of E1AF gene, anti-sense RNA of E1AF gene, an expression vector of the anti-sense RNA, a DNA binding domain of E1AF protein, a functional equivalent of a DNA binding domain of E1AF protein, an expression vector of a DNA binding domain of E1AF protein, an expression vector of a functional equivalent of a DNA binding domain oF E1AF protein, target DNA of a DNA binding domain of E1AF protein, an expression vector of target DNA of a DNA binding domain of E1AF protein, ribozyme for mRNA of E1AF gene, an expression vector of ribozyme for mRNA of E1AF gene, ribozyme for mRNA of a matrix metalloproteinase gene or an expression vector of ribozyme for mRNA of a matrix metalloproteinase gene can be used as a pharmaceutical composition for inhibiting cancer invasion which comprises as an effective component such a material.
Such a pharmaceutical composition for inhibiting cancer invasion can contain a pharmaceutically acceptable level of the anti-sense DNA, the anti-sense RNA, an expression vector of the anti-sense RNA, a DNA binding domain of E1AF protein, a functional equivalent of a DNA binding domain of E1AF protein, an expression vector of a DNA binding domain of E1AF protein, an expression vector of a functional equivalent of a DNA binding domain of E1AF protein, target DNA of a DNA binding domain of E1AF protein, an expression vector of target DNA of a DNA binding domain of E1AF protein, ribozyme for mRNA of E1AF gene, an expression vector of ribozyme for mRNA of E1AF gene, ribozyme for mRNA of a matrix metalloproteinase gene or an expression vector of ribozyme for mRNA of a matrix metalloproteinase gene, and can be prepared in the form of pharmaceutical preparations according to the similar manner as those for conventional therapeutic agents and gene-containing therapeutic agents. The pharmaceutical preparations can contain carriers, excipients, stabilizers, thickening agents and the like.
A dose of the pharmaceutical composition for inhibiting cancer invasion of the present invention can be selected by taking a patient""s state such as age, body weight, etc. and the degree of a diseased part into consideration.
The function of the anti-sense DNA, the anti-sense RNA or the expression vector of the anti-sense RNA to be contained in the pharmaceutical composition for inhibiting cancer invasion of the present invention is to inhibit expression of E1AF gene specifically. The function of a DNA binding domain of E1AF protein, a functional equivalent of a DNA binding domain of E1AF protein, an expression vector of a DNA binding domain of E1AF protein, an expression vector of a functional equivalent of a DNA binding domain of E1AF protein, target DNA of a DNA binding domain of E1AF protein or an expression vector of target DNA of a DNA binding domain of E1AF protein is to inhibit the matrix metalloproteinase-transcription-activating function of E1AF gene specifically. The function of ribozyme for mRNA of E1AF gene or an expression vector of ribozyme for mRNA of E1AF gene is to inhibit translation into E1AF protein. And, ribozyme to mRNA for a matrix metalloproteinase gene or an expression vector of ribozyme for mRNA of a matrix metalloproteinase gene is to inhibit translation into matrix metalloproteinase protein. Then, they are not toxic.
The pharmaceutical composition for inhibiting cancer invasion can inhibit metastasis of cancers which are highly metastatic, for example, can inhibit metastasis of fibrosarcoma.
In another aspect of the present invention, a method for detecting cancer is provided.
The detection of cancer can be accomplished by detecting a product expressed by E1AF gene represented by SEQ ID NO 5 of the Sequence Listing in tissue, after excising the tissue from a diseased part by an operation or biopsy. The detection of the expressed product can be carried out by preparing RNA from the isolated tissue and detecting mRNA of the expressed product of E1AF gene by northern hybridization, PCR or the like. A skilled artisan will readily design specific probes and primers to be used as probes in northern hybridization or as primers and probes in PCR based on the DNA sequence of E1AF gene as shown by SEQ ID NO 1 of the Sequence Listing.
In addition, cancer can be detected by detecting the expressed protein, E1AF protein, of the E1AF gene. For example, cancer can be detected by using the expressed protein, E1AF protein, and an immunoreactive antibody. The antibody can be prepared as a polyclonal or monoclonal antibody against E1AF protein. The antibody should be immunoreactive with an epitope of E1AF protein and, preferably, the epitope is not present in other proteins. In a preferred embodiment of the present invention, the antibody causes immunoprecipitation of E1AF protein in a solution and reacts with E1AF protein of western blotting or immunoblotting on polyacrylamide gel. In another preferred embodiment, the antibody can detect a protein in a paraffin embedded or frozen tissue section by using immunocytochemical techniques. The techniques for preparing and purifying an antibody are well known in this art and any of such known techniques can be used for producing an antibody preparation to be used in the present invention.
The method for detecting cancer of the present invention is applicable to any cancer wherein E1AF gene is expressed. That is, if the expression level of E1AF gene in tissue or cells is increased significantly over normal, such tissue or cells can be judged to be cancer tissue or cancer cells. Furthermore, malignancy of cancer can be judged by the expression level of E1AF gene. Examples of cancers to which the detection method of the present invention is applicable include fibrosarcoma, cancer of mammary gland, osteosarcoma, spontaneous transform endothelial cells, lung small cell carcinoma and the like.
In another aspect of the present invention, a method for diagnosing cancer tissue invasion is provided.
For diagnosing cancer tissue invasion, it is effective to isolate tissue which does not contain any surrounding normal tissue after excising the tissue by an operation or biopsy. A method for increasing cancer cell density in a tissue preparation has been known in this art. For example, paraffin embedded section or cryostat section can be used to isolate tissue. Cancer cells are also separated from normal cells by using flow cytometry. These and other techniques for separating cancer cells from normal cells are well known in the art. The presence of many normal cells in cancer tissue makes the diagnosis of cancer invasion difficult.
The diagnosis of cancer tissue invasion can be accomplished by detecting the product expressed by E1AF gene as shown by SEQ ID NO 5 of the Sequence Listing. The detection of the expression product can be carried out by preparing RNA from cancer tissue and detecting mRNA of the product expressed by E1AF gene by means of northern hybridization, PCR and the like. Further, cancer tissue invasiveness can be diagnosed by detecting E1AF protein expressed by E1AF gene. For example, the expressed protein, E1AF protein, in tissue can be detected by using an antibody which is immunoreactive with the expressed protein, E1AF protein. Such immunological analysis can be carried out by using a suitable known method including western blotting, immunohistochemical analysis, ELISA or the like.
The diagnostic method of the present invention can be applicable to any cancer wherein E1AF gene is expressed. That is, cancer tissue wherein the amount of the expression product of E1AF gene is increased significantly can be diagnosed to be highly invasive cancer tissue and, further, highly metastatic cancer tissue. Examples of cancers to which the diagnosis method of the present invention is applicable include fibrosarcoma, cancer of mammary gland, osteosarcoma, spontaneous transform endothelial cells, lung small cell carcinoma and the like. The diagnostic method of the present invention can be useful for determination of further treatment of a cancer-excised-patient by a clinician.
According to another aspect of the present invention, a kit for controlling cancer cell invasion comprising as a constituent component a material controlling the matrix metalloproteinase transcription-activating function of E1AF gene, for example, at least one component selected from the group consisting of E1AF protein, a functional equivalent of E1AF protein, E1AF gene, a gene encoding a functional equivalent of E1AF protein, anti-sense DNA of E1AF gene, anti-sense RNA of E1AF gene, an expression vector of anti-sense RNA of E1AF, a DNA binding domain of E1AF protein, a functional equivalent of a DNA binding domain of E1AF protein, a gene encoding a DNA binding domain of E1AF, a gene encoding a functional equivalent of a DNA binding domain of E1AF, an expression vector of a DNA binding domain of E1AF protein, an expression vector of a functional equivalent of a DNA binding domain of E1AF protein, target DNA of a DNA binding domain of E1AF protein, an expression vector of target DNA of a DNA binding domain of E1AF protein, ribozyme for mRNA of E1AF gene, an expression vector of ribozyme for mRNA of E1AF gene, ribozyme for mRNA of a matrix metalloproteinase gene and an expression vector of ribozyme for mRNA of a matrix metalloproteinase, as described above.
The material which controls the matrix metalloproteinase transcription-activating function, e.g., E1AF protein, in the kit may be either a solution or a lyophilized product. E1AF protein may be protein or its portion obtained by integrating E1AF gene represented by SEQ ID NO 1 of the Sequence Listing or a part thereof into an expression vector and expressing it. In so far as activating cancer cell invasion upon introduction into cells, any portion of E1AF protein can be used. In addition, the protein may be that extracted from E1AF protein producer cells. Moreover, the protein may be synthesized by synthetic chemistry. These proteins can be introduced into cells, maintaining their activities, by mircoinjection.
For example, E1AF gene may be in a solution or as a lyophilized product. E1AF gene represented by SEQ ID NO 1 of the Sequence Listing can be prepared by a method described in the above JP 5-328975 A or Nucleic Acids Research, 21, 547 553 (1993).
Storage conditions of the constituent components of the kit are not limited and, of course, can be suitably selected according to particular use of the kit and user""s convenience.
As described above, E1AF gene or a gene containing a part cut out from the gene, or a gene encoding a functional equivalent of E1AF protein or a gene containing a part cut out from the gene can be expressed in cancer cells efficiently by inserting the gene into an expression vector and introducing into the cells with the vector. When a gene containing a part of E1AF gene or a part of a gene encoding a functional equivalent of E1AF protein are introduced into cells and they are expressed, the part of the gene thus introduced should encode the necessary part for activating cancer invasion. As the expression vector, a vector for maintaining a gene extrachromosomally or a vector for integrating a gene intrachromosomally such as a retroviral vector can be used. In particular, cancer cells can be infected with a vector containing the gene efficiently by using a viral vector; As these vectors, retrovirus, vaccinia virus, adenovirus and a non-proliferative recombinant virus can also be used.
Anti-sense RNA for E1AF gene or a part of E1AF gene is expressed in cancer cells by inserting E1AF gene or a gene cut our from E1AF in the kit into a vector which is capable of expressing in cancer cells so that the anti-sense RNA of E1AF gene can be expressed and by introducing into cells with the vector. When a part of E1AF gene and the anti-sense RNA is expressed, in so far as the inserted part of the gene can inhibit the expression of cell endogenous E1AF gene and can express anti-sense RNA which can inhibit cell invasion, any part of the gene can be used. For example, cDNA corresponds to the 26th to 1783rd nucleotides and the 400th to 1118th nucleotides of SEQ ID NO 1 of the Sequence Listing can be used. As the expression vector of the anti-sense RNA, the same vector as described above with respect to the expression vector of E1AF gene can be used.
The kit for controlling cancer cell invasion of the present invention may contain as a constituent component synthesized anti-sense DNA or synthesized anti-sense RNA. In addition, the kit of the present invention can be combined with cancer cells, for example, human breast cancer cell line MCF-7, an in vitro invasion assay tool to constitute another kit. By using the kit for controlling cancer cell invasion of the present invention, cancer cell invasion can be readily controlled.
Furthermore, according to another aspect of the present invention, a kit for detecting cancer is provided.
This kit contains a materials for detecting a product expressed by E1AF gene. For example, the kit detects cancers by detecting mRNA expressed by E1AF gene and contains a probe which is hybridizable to mRNA of E1AF gene. One example of the probe is 1.56 kb E1AF cDNA represented by the 27th to 1586th nucleotides of SEQ ID NO 1 of the Sequence Listing. In so far as the probe is hybridizable to mRNA of E1AF gene specifically, any probe can be used and preferably a labelled probe is used. Further, mRNA of E1AF gene can be detected with high sensitivity by using a combination of an oligonucleotide primer for reverse transcription of mRNA of E1AF gene and a pair of primers for PCR of the reverse transcription DNA. Another example of the kit for detecting cancer is that detecting cancers by detecting E1AF protein expressed by E1AF gene and contains an antibody whose epitope is the expressed protein, E1AF protein, to detect the protein by immunological techniques.
Cancer cells, for example, fibrosarcoma, cancer of mammary gland, osteosarcoma, spontaneous transform endothelial cells, lung small cell carcinoma and the like produce a large amount of the expression product of E1AF gene in cancer cells and cancers in tissue can be therefore readily detected by using the kits for detecting cancers of the present invention. In addition, since these cancer cells produce a large amount of the expression product of E1AF gene and the amount of the production and cancer tissue invasion are correlated, the kit for detecting cancers can be used as a kit for diagnosing cancer tissue invasion. These kits may be solutions or lyophilized products. By using the kit for detecting cancers and diagnosing cancer tissue invasion of the present invention, it is possible to detect cancer in a diseased part and to diagnose malignancy of the cancer by utilizing cancer invasion as an index.
The following examples further illustrate the present invention in detail but are not to be construed to limit the scope of the present invention.